Impact of surface disinfection with hydrogen peroxide on the prevalence of vancomycin-resistant enterococci (VRE) in hospital wards

Objective: Vancomycin-resistant enterococci (VRE) are of major concern in infection control. Although broad infection control actions to check VRE have been implemented, VRE remain part of daily infection prevention in clinical settings. Cleaning procedures in the inanimate ward environment might play a key role in controlling VRE. In order to optimize infection control management at University Hospital Frankfurt, Germany (UHF), this study evaluates the impact of H2O2-containing cleaning wipes compared to Glucoprotamin containing wipes on VRE prevalence in intensive care wards. Methods: Retrospective analyses were conducted of the VRE prevalence on environmental materials obtained from three intensive care units (ICU) at UHF for 17 months prior to (T1) and during the 25 months after (T2) the implementation of H2O2-containing cleaning wipes from January 2016 to June 2019. The bactericidal power of the two disinfectants against VRE was compared using the 4-field test according to EN 16615 (2015). Results: At T1 and T2, n=666 and n=710 environmental samples, respectively, were obtained. At T1, 24.2% (n=161/666; 95% confidence interval: 21.0–27.6) and at T2, 6.9% (n=49/710; 5.1–9.0) samples were positive for VRE. In vitro disinfectant testing did not reveal any superiority of H2O2 over glucoprotamin. No effect on the VRE prevalence in patients’ rectal screening materials was observed. Conclusion: Though Glucoprotamin and H2O2 were in vitro equally effective against VRE, the prevalence of VRE in ICU environment at UHF decreased after implementation of H2O2-containig wipes. This might be due to multiple factors, of which we consider the impact of the Hawthorne effect to be the strongest. Success of infection control strategies might depend on the compliance of the persons critically involved. Transparent information on infection control strategies is suggested to increase compliance and should therefore be considered both in daily infection control and outbreak management.


Introduction
Vancomycin-resistant enterococci (VRE) are globally of major concern in terms of infection control and public health. Although broad infection control actions have been implemented, daily infection prevention measures must still deal with VRE. VRE are an important cause of morbidity and mortality and have been shown to increase the economic burden for hospitals when compared to vancomycin-susceptible isolates [1], [2], [3], [4], [5], [6]. In Europe, the proportion of invasive Enterococcus faecium isolates with resistance to vancomycin is inhomogeneous, with Germany ranking in the upper middle range [7]. Even within Germany, the proportion of VRE bloodstream infections varies greatly, with the highest percentages reported from the four federal states of North-Rhine Westphalia, Hesse, Thuringia and Saxony, which form the "VRE belt" [8], [9]. The epidemiology of VRE, however, is complex. This might be reflected by the increasing proportion of VRE in nosocomial infections due to enterococci as well as a gradual widening of the belt in Germany [8], [9], [10]. Although the reasons for this epidemiological phenomenon remain unclear, the National Public Health agency of France reports that glycopeptide resistant enterococci were most frequently reported from Ile-de-France, Lorraine and Nord-Pas-de-Calais [11], which might suggest that the belt stretches both over France and Germany. For the Rhine-Main area in Hesse, with almost three million inhabitants, the circulation of a single VRE clone (MLST type ST117, cgMLST complex type CT71 with a common vanB chromosomal insertion site) has been shown [12]. Located in the center of Hesse, University Hospital Frankfurt (UHF) has thus been facing one of the highest background VRE prevalences in Germany for several years. This is illustrated by the dramatically high number of cases (1,000 on average) annually of newly detected VRE in samples obtained from any patient body site. As the background prevalence remains unchangeable, the infection control strategy at UHF focuses intensely on the prevention of nosocomial VRE infection. UHF therefore offers a range of periodic and intensive tutorials, for instance addressing the epidemiology and current scientific status of multidrug-resistant organisms (MDRO), e.g., methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant gram-negative organisms (MDRGN), and VRE. These tutorials also address basic infection control strategies, e.g., hand hygiene, cleaning and disinfection procedures for patients' and wards' inanimate environments, which have been shown to play a key role in the spread of MDRO [13], [14], [15], [16], [17]. A small outbreak at UHF (five patients affected) of Klebsiella pneumoniae with carbapenem resistance in spring 2017 [18] impelled a change from glucoprotamin to H 2 O 2 -containing wipes for surface disinfection. This resulted in a continuous decline of VRE prevalence in material obtained from ICU environmental sites at UHF. Our findings therefore might encourage discussion of ways of fighting VRE in hospital settings.

Study setting and observation period
This study retrospectively investigated the VRE prevalence in near-patient inanimate environments in three wards at UHF before (T1) and after (T2) changing the wards' environmental cleaning procedure in June 2017. The observation period T1 included the 17 months from January 2016 to May 2017, and T2 included the subsequent 25 months until June 2019. Environmental specimens were taken by the same sampler over the entire observation period of 42 months.
Ward environmental cleaning procedure in T1 and T2 In June 2017, environmental cleaning procedures underwent a major change on three wards at UHF: one surgical ICU (ward 1), one internal medicine ICU (ward 2) and one surgical intermediate care unit (ward 3). This change was a result of outbreak management of K. pneumoniae with carbapenem resistance at UHF in spring 2017 [18]. Ward environmental cleaning procedures during T1 were performed using the antimicrobial disinfectant glucoprotamin (Incidin TM Plus Wipes, Ecolab, Monheim am Rhein, Germany) for surface disinfection. Since June 2017 (T2), ward environmental cleaning has been performed using a H 2 O 2 -based antimicrobial disinfectant (Incidin TM Oxy-Wipe S, Ecolab, Monheim am Rhein, Germany). During the observation period, no other new infection control interventions were implemented. The infection control protocol at UHF requires surfaces in patient rooms to be disinfected at least once daily and, in case of visible contamination, immediately. Furthermore, devices used daily, e.g. keyboards, computer mouse, ECG unit, need to be thoroughly wiped down with a disinfectant wipe after each use. Final disinfection of patients' rooms upon discharge or transfer of patients should include all surfaces, inventory, and equipment. Cleaning procedures are carried out by nursing and cleaning staff at UHF. Any positive VRE result is immediately reported to the ward's senior physician as well as to the ward's medical and nursing on-duty hygiene officer. They are informed of the location where VRE has been detected and are asked to carry out prompt disinfection of the respective device; they are also required to report this result at the ward's next team meeting and confirm proper execution of these actions as soon as it they have been performed.

Wards without permanent implementation of H 2 O 2 -based surface disinfection
In order to compare these findings with environmental samples obtained from wards on which the change from glucoprotamin to H 2 O 2 -containing wipes for surface disinfection was accomplished after than June 2017, we additionally evaluated the VRE prevalence in two wards which only temporarily changed to H 2 O 2 -based surface disinfection (ward N). These phases of temporary change to H 2 O 2 -containing wipes were 1. three weeks in November/December 2017 and 2. four weeks in March/April 2019.
Both episodes were due to non-availability of glucoprotamin at UHF. To analyze VRE prevalence after these episodes, the first sample round was taken and evaluated four weeks after the switch back to glucoprotamin. In case of positive VRE results, the same cascade described above was initiated.

Sampling and detection of VRE
In Germany, Infection Protection Law (Infektionsschutzgesetz; IfSG) determines several aspects of infection control, one of these being the mandatory epidemiological surveillance of organisms with multidrug resistance, such as VRE. As required by §23 IfSG [19], measures need to be taken in order to prevent the transmission of healthcareassociated pathogens, which also includes VRE. At UHF, this legal requirement is fulfilled inter alia by smear samples taken from inanimate environment, e.g., keyboards, drug-containing trolleys, medical devices or bandaging material, ECG units or ultrasound devices. The routine environmental smears are taken monthly from defined sites on the wards. All microbiological procedures were performed under quality-assured conditions (accredited standards according to ISO 17025:2005; certificate number D-PL-13102-01-00, valid through 2021) using cotton swabs (Süsse, Gudensberg, Germany) to take environmental smear samples, and were rubbed into tryptic soy broth plus lecithine/polysorbat 80 (Tween ® 80)/histidine/Nathiosulphate disinhibitor (LTHTh; Merck, Darmstadt, Germany). In case of turbidity within 48 hours of incubation at 36°C, the suspension was inoculated onto the CHROMID ® VRE plate (bioMérieux, Nürtingen, Germany) and incubated for a further 48 hours at 36°C. E. faecium were identified by matrix-assisted laser desorption ionisation -time of flight analysis (MALDI-TOF; VITEK MS; bioMérieux). Antibiotic susceptibility testing was performed according to international standard guidelines (Clinical and Laboratory Standards Institute) with VITEK 2 and antibiotic gradient tests (bioMérieux), as previously described [5].

VRE prevalence in patients' rectal screening samples
In addition, we retrospectively analyzed the prevalence of community-acquired (CA) VRE cases as well as VRE detected within the first three days after admittance, with the day of admittance being day one [20]; following the definition by the hospital-infection-surveillance system (Krankenhaus-Infektions-Surveillance-System, KISS) in accordance with the regulations of the National Reference Center for Surveillance of Nosocomial Infections, Berlin, Germany (Nationales Referenzzentrum für Surveillance von nosokomialen Infektionen). VRE cases detected beyond the first three days after admittance are categorized as "nosocomial detected (ND)". Since "nosocomial" has often mistakenly been supposed to be equivalent to "hospital-acquired", we recommend to use the terminus "nosocomial detected". The limitations of the current criteria to characterize "nosocomial" will be discussed below. Patients admitted to any ICU at UHF are routinely screened for VRE and other pathogens. Screening for VRE includes a rectal swab on the day of admission and repetitive VRE screening on a fixed-day weekly routine. This fixed-day weekly screening consists of all patients being treated on the ward on this specific day. This procedure is backed up by recommendations of the Commission for Hospital Hygiene and Infection Prevention (KRINKO) at the Robert Koch Institute on VRE [21], the weekly routine screening at UHF therefore even exceeds this. This evaluation is based on the results from rectal screening materials obtained from gastroenterological patients admitted to ward 2 in the 12 weeks prior to the outbreak of K. pneumoniae with carbapenem resistance at UHF in spring 2017 (T1; January 2017 to March 2017; [18]) as well as the 12 weeks following the completion of outbreak management (T2; June 2017 to August 2017). These patient groups and the periods were chosen for several reasons.
1. Ward 2 has the highest patient VRE prevalences at UHF, which predetermines it to observe any VRE prevalence changes, however minor. 2. The gastroenterological patients admitted to ward 2 are similar in terms of medical treatment: they are all likely to present after a long history of medical pretreatment, which implies similar exposure to antibiotic (selective) pressure in their medical history. 3. We therefore assume that the VRE prevalence in patient screening materials has largely been stable.
In order to estimate the patients' VRE prevalence in ward 2, an observation period of 12 weeks seems to be appropriate to approach the patients' base VRE prevalence. In order to assess the patients' VRE prevalence in the H 2 O 2 cleaning area, the observation period must be started as soon as possible after completion of the outbreak management. In order to exclude any over-or un- 2), respectively. Further details are shown in Figure 1.

VRE prevalence in wards without permanent change to H 2 O 2 -containing cleaning wipes
As illustrated in Figure 3, VRE prevalence in theses wards' environmental samples amounted to an average of 23

Results of disinfectant testing
The results obtained at 2 or 5 min time of action are presented in Table 1. The strains (a), (b) and (c) were as mentioned above.
The data indicate that there was no difference regarding the test product (glucoprotamin vs. H 2 O 2 ), the test organism (strain 1, 2 or 3) or the time of action (2 vs. 5 min).
All the controls were valid as demanded by EN 16615. However, even if differences may exist at lower concentrations of the test products, none were observed when the products were tested under conditions quite similar to their application on UHF wards.

Discussion
The German VRE belt is formed by North-Rhine Westfalia, Hesse, Thuringia and Saxony [8], [9], [10]. Thus, University Hospital Frankfurt is centrally located in this area of high background VRE prevalence and, as a result of this exposure, faces almost n=1,000 of newly detected VRE cases per year in screening and clinical samples obtained from patients admitted to UHF. As part of daily routine infection control, VRE is therefore continually addressed in hygiene and cleaning training courses as well as seminars for employees at UHF. Despite the continuous efforts of all staff, the VRE prevalence in environment material was ca. 24% at UHF between January 2016 and May 2017 (T1). In June 2017, however, the environmental disinfectant procedure changed from glucoprotamin to H 2 O 2 -containing cleaning wipes in three intensive care units (Figure 1), which resulted from the management of an outbreak at UHF [18]. Afterwards, the VRE prevalence in environmental material sharply declined to 8.1% in 2018 and 2.6% in 2019 (as of 30 June 2019; Figure 2). This was also observed for wards which had only temporarily changed from glucoprotamin to H 2 O 2 -containing cleaning wipes ( Figure 3). This dynamic is remarkable, since H 2 O 2 has not proven to be superior to glucoprotamin in terms of efficacy against VRE. Therefore, one or possibly a combination of factors is suggested to be responsible for this trend. In the phase immediately following the change and in the aftermath of outbreak management at UHF in spring 2017 [18], we propose that the observed decline of VRE prevalence was due to the Hawthorne effect, which might have led to behavioral changes among staff involved in infection control interventions and might thus have encouraged this trend [25], [26], [27]. As shown in other infection control studies, however, this effect is only temporary [27], [28], [29], indicating that the VRE prevalence in environmental samples at UHF might be due to additional factors. One of these might be the practical handling of glucoprotamin compared to H 2 O 2 -containing cleaning wipes. Glucoprotamin-containing wipes are not supplied in a ready-touse form. Before being operational, the dry wipes need to be unpacked and placed in a dispenser. Then, 2.5 liters of Incidin TM disinfection solution must be poured into the dispenser and the first wipe needs pulled through the dispenser's lid. After closing the dispenser, the disinfection solution must be allowed to be absorbed by the wipes. Only when wipes are completely saturated are they ready to use, which takes up to 60 minutes (manufacturer's information) [30]. In contrast, the H 2 O 2 -containing wipes are supplied pre-assembled, saturated and ready for immediate use, which is more user-friendly [29], [31]. Furthermore, subjective sensations might additionally contribute to this trend. Whereas the scent of glucoprotamin-containing wipes is almost neutral, the scent of H 2 O 2 wipes is distinctive and might be characterized by some as acrid. This subjective sensation might have increased the users' compliance [32] -similar to the phenomenon that the perceived effect of drugs depends on their color [33], [34]. These two effects are supposed to buttress the Hawthorne effect and might therefore have additionally contributed to the decline of the VRE prevalence in the environmental samples throughout the entire period from June 2017 to June 2019 (T2  Figure 1 and Figure 2), but this specific outbreak-related training is highly unlikely to have affected the VRE prevalence in 2018 and 2019. The findings of this study might be limited by several factors. First, it was a single-center study and the results therefore can only be transferred to other hospitals to a limited extent (e.g., due to different local VRE prevalence, local screening procedures and medical profile of the institution). In particular, the profile of university medicine is clearly not comparable to the medical profile of a hospital providing only basic health care; university hospitals admit a high number of special-needs patients who are more likely to carry a MDRO, compared to the clientele of hospitals with a lower medical profile. Because at UHF we take swift infection-control action any time VRE are detected on environmental surfaces, independent of the number of colony forming units found, in this study, we did not determine the quantity of VRE in the environmental samples. Admittedly, this might have been necessary to more reliably quantify VRE contamination. In future studies, this aspect should be evaluated. Regarding patients' CA-VRE:ND-VRE ratio on ward 2, no effect was detected after the implementation of H 2 O 2 -containing cleaning wipes. However, this was not surprising, since the background prevalence of VRE is consistently high in the Rhine-Main region [8], [9], [10] and will not be affected by our change in ward cleaning management. To estimate any effect on the number of nosocomial cases, the data of all patients -those who had only one screening in particular -would be needed. In addition, data on the sensitivity of one single rectal screening for VRE are lacking up to now. Hence, it remains unclear whether a patient who screened negative upon admission to the hospital but in a subsequent positive screening after submission must be classified as "nosocomially detected" or as a slipped case in initial screening. These aspects should be addressed in further research, in which the medical profile of university hospitals should also be considered, as this is where patients with complex conditions, often protracted clinical histories after pretreatment in various other hospitals, and long histories of antibiotic treatment are frequently admitted. This, however, makes obvious the limitations of the current criteria for defining "nosocomial" as recommended by KISS and Centers for Disease Control and Prevention (CDC), as previously discussed [39]. Currently, "nosocomial" is defined in terms of time of the pathogen's first detection with regard to the day of admittance. In case of the occurrence of a "resistance plasmid transfer" [40], the label "nosocomial" can also be highly problematic, as the transfer of a resistant plasmid from one species to another, e.g., VanA from VRE to Staphylococcus aureus, resulting in vancomycin-resistant S. aureus [40], might be misinterpreted as a new nosocomial acquisition. The prevalence of VRE in environmental materials has significantly decreased since switching from glucoprotamin to H 2 O 2 -containing cleaning wipes at UHF. Although H 2 O 2 and glucoprotamin had equivalent efficacy against VRE, several other factors are supposed to be responsible.
In conclusion, the initial effect might have been caused by a raised general awareness for infection control as a result of an outbreak managed at UHF in spring 2017 [18]. Since any positive VRE result is quickly reported to key players on the ward and prompt actions are required, the Hawthorne effect is suggested to have stabilized the adherence to infection control throughout the entire observation period. Thus, attenuation of the Hawthorne effect in the UHF setting seems to be unlikely. Handling or subjective perception, e.g., scent, might have also contributed to this effect. Additionally, increased self-efficacy among the staff might also have contributed to this trend. Based on our experience, adherence might be strengthened by transparently providing information on the sense and purpose of infection control strategies. We therefore suggest that this should be considered in both daily infection control routines and outbreak management.

Notes
Competing interests